Signaling from G protein-coupled chemoattractant receptors to the functional responses of human leukocytes involves Rho small GTPases. In previous studies, we established that p21-activated kinases (PAKs) are important downstream mediators of Rho GTPase signaling, and regulate crosstalk between the actin- and microtubule-cytoskeletons. We identified molecular targets for cytoskeletal regulation by PAK1, including GEF-H1, LIM kinase, and myosin light chain kinase. In the current proposal, we will investigate the action of PAK1, as well as its target GEF-H1 in cytoskeletal regulation using biochemical, genetic, and molecular approaches. We will continue studies of the spatio-temporal dynamics of Rho GTPase activation and function during chemotaxis using unique fluorescence-based imaging technologies. We have characterized GEF-H1 as a microtubule-regulated guanine nucleotide exchange factor for Rho GTPase, and implicated GEF-H1 in the regulation of Rho activity during cytokinesis and directional cell motility. The biological activities of GEF-H1 will be investigated using genetic and siRNA-based approaches. The role(s) of PAK1 in modulating GEF-H1 function will be studied. We have identified a role for PAK1 in regulating retrograde actin flow (RAF), a dynamic process important for cell motility. We will use quantitative fluorescence speckle microscopy (FSM) combined with genetic and biochemical approaches to define the downstream mediators of PAK1 action in RAF. Finally, FRET-based methods for visualizing Rac and Rho activation will be used to analyze the spatial and temporal dynamics of these GTPases in the chemotactically responding human neutrophil. The role of Rac2 in uropod retraction will be studied at the biochemical level. Spatio-temporal evaluation of Rho activation will be related to Rac2 activity, as well as specific aspects of a) chemoattractant receptor signaling, b) motile behavior, and c) actin-myosin cytoskeletal dynamics.